Association between MTHFR (677C>T and 1298A>C) polymorphisms and psychiatric disorder: A meta-analysis

Recent studies showed that genetic polymorphism of 5,10-methylenetetrahydrofolate reductase (MTHFR) is related to attention-deficit hyperactivity disorder (ADHD), bipolar disorder (BD) and schizophrenia (SCZ). However, no consistent conclusion has been determined. This meta-analysis aims to interrogate the relationship between MTHFR gene polymorphisms (677C>T and 1298A>C) and the occurrence of ADHD, BD and SCZ. We retrieved case-control studies that met the inclusion criteria from the PubMed database. Associations between MTHFR polymorphisms (677C>T and 1298A>C) and ADHD, BD and SCZ were measured by means of odds ratios (ORs) using a random effects model and 95% confidence intervals (CIs). Additionally, sensitivity analysis and publication bias were performed. After inclusion criteria were met, a total of five studies with ADHD including 434 cases and 670 controls, 18 studies with BD including 4167 cases and 5901 controls and 44 studies with SCZ including 16,098 cases and 19913 controls were finally included in our meta-analysis. Overall, our meta-analytical results provided evidence that the MTHFR 677C>T was associated with occurrence of BD and SCZ, while the 1298A>C polymorphism was related to ADHD and BD, and additionally the sensitivity analysis indicated these results were stable and reliable. This may provide useful information for relevant studies on the etiology of psychiatric disorders.


Introduction
Folic acid, a member of the vitamin B complex, in considered to be strongly associated with the function and development of the central nervous system, which plays an important role in cellular processes including nucleotide synthesis and methylation [1]. The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) functions in the pathway that converts folate into metabolites that may be used for cellular processes including methylation of gene promoter enhancers and protein, RNA, DNA, amino acid and phospholipid synthesis. Specifically, this enzyme converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which (sample size 29,502) [33]. There have been four meta-analyses concerning the association of SCZ [33][34][35][36]. The latest study found that MTHFR A1298C polymorphism was a risk factor for SCZ, which included 19 studies with 4049 cases and 5488 controls [36]. To better understand the role of MTHFR in the occurrence of psychiatric disorders, we conducted a meta-analysis of all published case-control studies exploring the associations between two common polymorphisms (677C>T and 1298A>C) of MTHFR and three psychiatric disorders: ADHD, BD and SCZ. This will provide a more comprehensive assessment of the association between this polymorphism and ADHD, BD and SCZ.

Identification and eligibility of relevant studies
To identify eligible studies for inclusion in this meta-analysis, we searched the PubMed electronic database up to December 2021, without restriction on article type in English. The following keywords were used in the literature search: 5,10-methylenetetrahydrofolate reductase, MTHFR, and one of the following three words: ADHD, BD or SCZ. The selected studies met the following inclusion criteria: (1) case-control design, (2) including patients with one of the three diseases and (3) stating available allele or genotype frequencies. Of the studies with the same or overlapping data published by the same authors, the latest articles were selected. Major reasons for exclusion follow: (1) no control population, (2) duplicate of an earlier publication and (3) lack of usable genotype frequency data. If we needed to retrieve additional data that were not contained in the original report, we contacted the corresponding authors for additional details (e.g., allele or genotype frequencies or sample characteristics).

Data extraction
Based on the inclusion criteria, two reviewers (Mao-ling Sun and Jun Yao) independently extracted information from all the included studies. Disagreements were resolved by discussion until the two reviewers reached a consensus. The following data were extracted from each study: first author's family name, publication year, country and number of genotypes between cases and controls. To delineate potential moderating influences on the effects obtained from the case-control studies considered, we also included the following variables: (1) diagnostic criteria, (2) controls source, (3) mean age of cases and (4) proportion of males in the disease sample.
studies. The degree of heterogeneity between studies was determined by Q-statistic, with p > 0.05 indicating a lack of heterogeneity and p < 0.05 indicating heterogeneity. Moreover, I 2 was calculated to quantify the apparent inconsistency; its conventional interpretation for existing heterogeneity is low (<25%), moderate (approximately 50%) and high (>75%). Additionally, Begg's funnel plot and Egger's test were used to evaluate publication bias. Sensitivity analysis was performed to assess the potential influences of a single study on the pooled effect size. It was performed by omitting single studies one at a time for each meta-analysis to screen for significant alterations to pooled effect size.
All statistical tests were two-sided, with p < 0.05 considered significant. The meta-analysis was conducted using Stata version 16.0 software (Stata Corp., College Station, TX, USA).

Results
After the removal of overlapping articles and those that did not meet the inclusion criteria ( Fig  1), a total of five studies with ADHD including 434 cases and 670 controls [2,[37][38][39][40]

Sensitivity analysis
We examined the influence of individual studies in the pooled ORs for 667C>T and 1298A>C loci via sensitivity analysis involving omitting each study in each genetic model; the results did not change. This indicates that our results were statistically robust for all five genetic PLOS ONE models examining associations between MTHFR polymorphisms and susceptibility to ADHD, BD and SCZ.

Publication bias
We assessed possible publication bias using a Begg's funnel plot and Egger's test. No obvious asymmetry was observed in the funnel plot and Begg's test results, indicating a lack of publication bias (p > 0.05) except for the homozygous codominant model of 677C>T locus in BD (p = 0.025) (Figs 8-13).

Discussion
The present meta-analysis included 66 studies that investigated the association between MTHFR (677C>T and 1298A>C) polymorphisms and occurrence of ADHD, BD and SCZ. Overall, our meta-analytical results provided evidence that MTHFR 677C>T was associated with occurrence of BD and SCZ, while the 1298A>C polymorphism was related to ADHD and BD. The sensitivity analysis indicated that these results were stable and reliable. Five previous retrospective studies investigated the association between MTHFR polymorphisms and ADHD [2,[37][38][39]77]. Our results were very similar to those of Tahereh Sadeghiyeh [77], but not exactly the same as those of Saliha Baykal and Emel Ergul [37,38]. A total of five retrospective studies were included, which represented MTHFR polymorphisms more accurately than previous published studies. This is the first meta-analysis to include recent published studies concerning the association between MTHFR polymorphism and ADHD occurrence. Therefore, to some extent, our study provides a more reliable assessment of the association between MTHFR polymorphisms and ADHD. Additionally, some previous studies showed that ADHD occurrence was affected by various environmental factors [78]. It is possible that epigenetic risk mechanisms in ADHD responding to environmental risk factors or trans-regulatory and gene × environment effects in the development of child psychopathology might play a consequential role in ADHD etiology [79]. In addition, ADHD subtypes represent distinct clinical entities and may have different genetic backgrounds [80]. To date, case-control studies and meta-analyses have explored the role of MTHFR polymorphisms in BD occurrence [24,31,33,43,51,[81][82][83] but with no consistent conclusion. Additionally, The MTHFR gene polymorphism is unlikely to play a major role in the pathogenesis of obsessive-compulsive disorder [84]. Our study showed that the 677C>T and 1298A>C polymorphisms were involved in the occurrence of BD. Moreover, a genome-wide association study suggested that the MTHFR gene polymorphism was related to mood disorder [85]. The Genotypes of 677C>T were related to total homocysteine (tHcy), folate and B12. Individuals with TT genotype have elevated tHcy and reduced folate and B12 levels, which may be a susceptible factor for the BD [48]. The interaction of BDNF Val66Met and MTHFR C677T may reduce the hippocampal size in both healthy controls and patients with first-episode psychosis [86].
The C677T polymorphisms of MTHFR had an influence on SCZ symptoms. However, the effect of the T allele on the negative symptoms of SCZ could be further enhanced by folate deficiency [87]. Additionally, there was a significant association between the 677TT genotype and SCZ under the recessive model in the male patient subgroup, and CT genotype under the overdominant model in the total patient group [65]. The OR for patient with BD and SCZ in 1298CC homozygous state was 3.768 (P = 0.0003) and 2.694 (P = 0.0123), respectively. After the stratification of patients based on gender, only a significant association of 1298CC genotype with BD in female patients was observed (P = 0.0005) [46]. Moreover, a previous metaanalysis indicated that the T allele and TT genotype of C677T carriers showed significantly increased risk of major psychiatric disorders including SCZ and BD [33]. Moreover, the activity of MTHFR will be affected by multiple single-nucleotide polymorphisms. However, variations other than the 677C>T and 1298A>C polymorphisms have received little attention. In addition, aggravating symptoms, increased MTHFR polymorphisms, and reduced genomic methylation levels can be observed in patients with early-onset SCZ [88]. MTHFR 677T allele carriers have lower levels of total cholesterol and low-density lipoprotein cholesterol than those with the 677CC genotype [89]. There was a positive association between the COMT-MTHFR interaction and attention in inpatients suffering from recent onset SCZ [90]. MTHFR A1298C, but not C677T, was associated with the metabolic syndrome, its CC genotype having a 2.4 times higher risk compared to AA genotype [91]. In addition, the C allele of MTHFR was associated with BMI reduction in the schizophrenia patients following switching of antipsychotics to aripiprazole and ziprasidone [92].
There were several potential limitations to the present study. First, the most important was sample size. Small samples with limited participants are usually accompanied by selection biases. These studies lack sufficient power to support or refute meaningful conclusions [93]. Second, subgroup analysis cannot be carried out with limited samples, so the influence of some factors (e.g. ethnicity, source of controls and diagnostic criteria) were ignored. The discrepancies of the studies may result from population stratifications, explicitly, socio-economic status [94]. Finally, clinical subtypes of the mental disorder, gene-gene interaction and epigenetics were not examined in this study due to insufficient information.

Conclusions
Our findings suggest that the MTHFR 677C>T was associated with occurrence of BD and SCZ, while the 1298A>C polymorphism was related to ADHD and BD. Studies involving larger sample sizes will be necessary to confirm the meta-analysis results, particularly in different ethnicities and to address the epigenetic mechanisms and environmental influences on the occurrence of common mental disorders.